Preparation of (-)-nutlin-3 using enantioselective organocatalysis at decagram scale

Article abstract of DOI:10.1021/jo401321a

Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more general use. Our disclosure of the first enantioselective synthesis of (-)-Nutlin-3 provided a basis to prepare larger amounts of this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (-20 C) and low catalyst loadings. Further refinements to the procedure led to the synthesis of (-)-Nutlin-3 in a 17 g batch and elimination of all but three chromatographic purifications.

Full text of DOI:10.1021/jo401321a

Article
pubs.acs.org/joc
Preparation of (−)-Nutlin‑3 Using Enantioselective Organocatalysis at
Decagram Scale
Tyler A. Davis,‡,† Anna E. Vilgelm,§ Ann Richmond,§ and Jeffrey N. Johnston*,‡
‡Department of Chemistry & Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235-1822,
United States
§Department of Veterans Affairs, Tennessee Valley Healthcare System, and Department of Cancer Biology, Vanderbilt School of
Medicine, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of
cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-
type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more
general use. Our disclosure of the first enantioselective synthesis of (−)-Nutlin-3 provided a basis to prepare larger amounts of
this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel
bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (−20 °C) and low catalyst
loadings. Further refinements to the procedure led to the synthesis of (−)-Nutlin-3 in a 17 g batch and elimination of all but
three chromatographic purifications.
patents4 and two reports in late 20129 can be used to prepare
INTRODUCTION
■
Nutlin-3 in racemic form, but the need for supercritical fluid
The development of small-molecule inhibitors of p53/MDM2
chromatography to obtain the more potent enantiomer10
has grown rapidly1 in the intervening years since the 2004
diminishes its general access as well.
We developed a highly diastereo- and enantioselective
discovery by scientists at Hoffmann-La Roche (HLR) that a
new class of cis-4,5-disubstituted imidazolines were effective
inhibitors of this protein−protein interaction.2 Among these
addition of aryl nitromethanes to imines and applied the
reaction to the first synthesis selective for (−)-Nutlin-3.3 A key
heterocycles, Nutlin-3 was the most potent derivative, and the
advantage of this approach is the access it provides to
levorotatory enantiomer (1) is now known to be the more
potent antipode.2−4 The HLR discovery fueled countless
desymmetrized cis-stilbene diamines in two steps through a
carbon−carbon bond-forming reaction. Seidel’s desymmetriza-
tion of cis-stilbene diamines using an enantioselective acylation
studies in basic cancer biology, stimulated the discovery of
similar small molecule inhibitors by others,1,5 and provided
optimism in cancer drug development.6 HLR recently advanced
is a promising alternative strategy and may ultimately provide
one member of this class to phase I clinical trials.7
Studies that use (−)-Nutlin 3 as a tool to elucidate basic
pathways in cancer cell biology must ultimately be validated in
vivo. A typical experiment in a mouse model requires 500 mg of
(−)-Nutlin-3, an amount that would require approximately US
$15000 if provided by a commercial source.8 Although this cost
represents a discount of 50% since our report in 2011, it
remains a significant financial obstacle to most biology
laboratories. Alternatively, the HLR synthesis reported in
an enantioselective route from the cis-stilbene diamine common
to the HLR synthesis.11
Although the aza-Henry approach appeared to be advanta-
geous by both strategic and practical criteria, we sought to
examine several factors critical to validation of the route’s
suitability for large scale implementation, including: (1)
Received: June 25, 2013
Published: October 15, 2013
© 2013 American Chemical Society
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optimization of the key enantioselective step (catalyst loading
and temperature) and (2) minimization or elimination of
chromatography steps. The result of this study is a batch
procedure to produce up to 17 g of (−)-Nutlin 3 and a new
catalyst that allows the key enantioselective step to operate at
−20 °C with high enantioselectivity.12
Hoffmann-La Roche patents outline their use of meso-
diamine 2 (synthesized in five steps from benzil, Scheme 1).
Figure 1. Building blocks required for the enantioselective synthesis of
(−)-Nutlin 3.
Scheme 1. Hoffmann−La Roche Synthesis of rac-Nutlin-3
coupling.20 A recent advance, reported after completion of our
study, is that of Kozlowski,21 who described the cross-coupling
of aryl halides and nitromethane to give the desired aryl
nitroalkanes in high yield.
In our hands, Kornblum’s procedure was superior. The
arylnitroalkane is prepared in two steps from p-chlorotoluene
(6) with this method, although p-chlorobenzyl bromide 7 is
commercially available. Treatment of p-chlorotoluene with N-
bromosuccinimide and a substoichiometric amount of AIBN
provided the desired benzyl bromide in 78% yield (127 g) after
crystallization.
This benzyl bromide (7) was then treated with sodium
nitrite to provide the desired nitroalkane 8. Initially, a
procedure using a nitrite resin described by Gelbard22 was
attempted, and although the straightforward purification by
filtration was advantageous, we were unable to achieve the yield
(87%) described for this substrate.
Application of the Kornblum procedure was particularly
sensitive to the reaction time, as excessive time resulted in an
increased ratio of benzyl alcohol to nitroalkane. Benzyl alcohol
likely results from nitrite alkylation at oxygen (forming benzyl
nitrite), followed by nitrosyl transfer to an incidental
nucleophile (e.g., water, silica gel). This nitrosyl transfer is
necessary to prevent a side reaction between benzyl nitrite and
the desired nitroalkane 8 which reduces yield. Phloroglucinol, a
nitrite ester trapping agent,15 was not effective in converting the
nitrite ester to benzyl alcohol when used as a component of the
reaction; it was not until workup that this conversion occurred.
Therefore, phloroglucinol was added to the ethereal solution
after an aqueous workup. Within minutes, the slightly golden
solution became the dark red color reportedly characteristic of
nitrosophloroglucinol. This solution was washed with water,
dried, and concentrated. The residue could not be purified by
vacuum distillation, acid/base extraction, or recrystallization,
but purification by column chromatography provided 27.8 g
(42%) of the nitroalkane 8.
The N-Boc imine 12 (Scheme 2) is made in two steps that
involve simple purification techniques from readily available
starting materials. The N-Boc α-amido sulfone23 11 is made
from the condensation of the corresponding aldehyde 9, N-Boc
tert-butyl carbamate 10, and the sodium salt of phenylsulfinic
acid in the presence of formic acid.24 Synthesis of this particular
sulfone has been described twice in the literature by Wulff, with
yields of 23% (3 days reaction time, 1.2 equiv of aldehyde)25
and 61% (2 days reaction time, 2 equiv of aldehyde).26 Similar
conditions in our hands proved reasonably general across a
wide range of sulfones. In an attempt to obtain higher yields
through increased conversion, we lengthened the reaction time
to 9 days before the precipitated product was filtered.
Trituration of the filtered solid with ether removed residual
aldehyde and provided 53.7 g of sulfone 11 (66% yield). In an
Compound 2 could be cyclized with amidate 313 or methyl
ester 4a in the presence of AlMe314 to provide meso-imidazoline
5a/5b. Treatment of 5a/5b with phosgene gave a mixture of
acid chloride enantiomers which were separated by chiral
HPLC before or after substitution of the chloride with the
amine to give (−)-Nutlin-3 and derivatives. Details related to
HLR’s process development of Nutlin-3 have emerged
recently,9 describing a boric acid catalyzed cyclization to form
imidazoline intermediate 5b. While this is a significant
improvement, the approach provides Nutlin-3 in racemic
form and relies upon supercritical fluid chromatography to
separate the desired enantiomer in the final step.10
RESULTS AND DISCUSSION
■
Our approach to Nutlin-3 is based upon four key building
blocks as illustrated in Figure 1. In order to scale this synthesis,
each building block needed to be accessible in significant
quantity.
Several methods were available to prepare the aryl nitro-
methane (8, Scheme 2). The prevailing method is displacement
of the corresponding bromide with nitrite.15 This preparation is
often characterized by low yields due to competing O-
alkylation, but it is reliably applied and a collection of diverse
benzyl halides are commercially available. Alternative prepara-
tions include electrophilic nitration of the corresponding
metalated toluene,16,17 electrophilic nitration and concomitant
decarboxylation of phenylacetonitrile or phenyl acetic acid,18
free radical arylation of nitromethane,19 and Pd-catalyzed
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a
Scheme 2. Preparation of the Synthetic Building Blocks
a
Key: (a) NBS, AIBN, CCl4, reflux; (b) NaNO2, urea, DMF,−20 °C; (c) NaSO2Ph, HCO2H, MeOH/H2O, rt, 19 d; (d) K2CO3, Na2SO4, THF,
i
reflux; (e) K2CO3, PrBr, DMF, reflux; (f) KOH, EtOH/H2O, reflux.
attempt to further increase yield, we combined the trituration
filtrate with the original filtrate, stirred the mixture in an open
vessel for an additional 10 days, and collected the nascent
precipitate. After trituration, an additional 15.3 g of sulfone was
isolated. The total yield for this reaction on the largest scale
attempted was 68.9 g after 19 days (85%). Attempts to further
improve the reaction rate (e.g., temperature increase, solvent
mixtures) were unsuccessful. The simplicity of the reaction
conditions, ease of purification, and high yield provided an
adequate platform to launch a large-scale synthesis.
Formation of the N-Boc imine (12) by treatment of the α-
amido sulfone with potassium carbonate and sodium sulfate in
refluxing THF was dependent on reaction time. Reactions that
were stopped too long after full conversion contained aldehyde
(the result of imine hydrolysis), while (prematurely stopped)
reactions containing unreacted α-amido sulfone could produce
benzene sulfinate (or its conjugate acid) during the subsequent
aza-Henry step. The latter was expected to work against the
goal to lower catalyst loadings, as overprotonation of the
catalyst can adversely affect reactivity and/or stereoselection.27
As a result, careful monitoring of imine formation was applied,
and on the largest scale attempted, this reaction provided 15.2 g
of the N-Boc imine 12 (95%) after a 6 h reaction period. The
isolated material contained ∼4% of the parent aldehyde.
The requisite carboxylic acid 15 was synthesized in two steps
from commercially available 4-methoxysalicylic acid (13,
Scheme 2). The dialkylated product 14 was produced in 93%
yield (13.2 g) after purification. Subsequent hydrolysis of the
dialkylated compound provided the desired carboxylic acid 15
in excellent yield (94%, 10.4 g).28
Our initial rendition of the enantioselective aza-Henry
Figure 2. Aryl nitromethane additions at low temperature. All
reaction employed 5 mol % 6,7(MeO)2PBAM (18, Figure 2),
reactions used 0.1 mmol imine and 1.1 equiv of nitroalkane in 0.1
low temperature (−78 °C), and chromatographic purification
M toluene
of the desired product. In order to adapt the procedure to a
large-scale preparation, we sought a protocol that could be
applied at higher temperature, and a purification technique that
did not involve chromatography. Novel catalyst modifications
were explored to achieve the high selectivity at warmer
operating temperatures, while direct precipitation was used to
streamline the overall protocol.
The relative insolubility of the major diastereomer of the aza-
Henry product under the initial conditions proved to be
beneficial both in the optimization of this reaction and the
scale-up of the (−)-Nutlin-3 synthesis overall. The product
precipitated from solution during the reaction at more
concentrated conditions (≥0.1 M, toluene). This allowed for
a simple filtration of the reaction to isolate the desired
diastereomer in high purity. This protocol was therefore used
to facilitate an examination of reaction variables and ultimately
identification of the optimal conditions. As one would
anticipate, increasing the reaction temperature leads to an
attenuation of stereoselection. Identification of a more selective
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Scheme 3. Preparation of the Pyrrolidine Bis(amidine) Catalyst 19
a
catalyst that could operate at higher temperature became an
important objective.
Table 1. Aryl Nitromethane Additions at −20 °C
When using aryl nitromethane pronucleophiles, enantiose-
lectivity observed when using 4-pyrrolidine bisamidine (PBAM,
17)29 is not improved by the addition of an equal amount of
triflic acid, as is often the case with most other nitroalkanes.30,31
Our hypothesis is that the acidic aryl nitroalkane serves as the
Brønsted acid cocatalyst in these reactions.32−34 The
assumption that the more basic 4-pyrrolidine catalysts are the
best catalysts for this reaction led us to retain this element in
catalyst design efforts. In one investigational line, an increase in
the catalyst Brønsted basicity was sought by catalysts with
electron-releasing quinoline substituents, while positioning
b
b
c
entry
catalyst
dr
ee (%)
yield (%)
1
2
3
PBAM
17
18
19
6:1
6:1
7:1
83
80
89
99
91
99
6,7(MeO)2PBAM
8MeOPBAM
these groups proximal to the site of presumed substrate
binding. This ultimately resulted in the identification of
a
0.1 mmol scale, 1.1 equiv of 8, 0.1 M in toluene, 18−24 h reaction
time. Diastereomer and enantiomer ratios measured by HPLC using
b
6,7(MeO)2PBAM (18) as the most enantioselective catalyst.
Despite the high enantioselectivity produced by this catalyst
(91% ee), these values could only be achieved at −78 °C.
This prompted a further investigation of catalyst modifica-
tions in order to achieve high enantioselectivity, and one aim
was to explore substitution at the 8-position. Of the positions
5−8, the 8 position was expected to be the position of highest
steric consequence due to its proximity to the amidine
functional group. While our operational stereochemical models
presume amidine protonation, followed by imine binding to the
amidinium, it was not clear whether substitution at the 7- or 8-
position would provide improved facial bias or hindered
substrate binding. These bis(amidine) variations could be
c
chiral stationary phases. Isolated yield.
a
Table 2. Catalyst Loading Comparison Of Two Catalysts
PBAM (17)
6,8(MeO)2PBAM (23)
prepared using our standard approach, and illustrated for 19
b
c
b
c
ee
yield
(%)
ee
yield
(%)
8
(Scheme 3). An initial comparison of the free base of
MeOPBAM (19) to PBAM (17) and 6,7(MeO)2PBAM (18)
(Figure 2) under the previously optimal conditions revealed the
8-substituted catalyst to be significantly more enantioselective,
providing the product in 9:1 dr and 95% ee at −78 °C without
a noticeable drop in reactivity. The same trend was observed
with these three catalysts when warmer conditions were used
entry mol %
(%)
entry mol %
(%)
1
2
3
4
5
6
20
10
5
53
65
70
73
74
80
39
59
71
76
76
78
1
2
3
4
5
6
7
15.4
7.7
62
73
79
84
86
87
87
58
68
75
74
79
79
73
3.9
2
1.5
1
0.77
d
1
0.38
0.15
8
(−20 °C, Table 1). Once again, MeOPBAM (19) was found
a
to be the most enantioselective catalyst of the three giving
appreciably high enantioselectivity (89% ee).
0.1 mmol scale, 1.1 equiv of 8, 0.1 M in toluene, 18−24 h reaction
time. Determined by chiral HPLC. Isolated yield of single
diastereomer (200:1 dr by HPLC) after filtration/washing. 0.2
equiv of imine added, 30 min intervals.
b
c
d
The relatively high reactivity of the aryl nitroalkanes
compared to aliphatic nitroalkanes with the 4-pyrrolidine
bis(amidine) class of catalysts35 offered the potential to utilize
very short reaction times and/or low catalyst loadings. With
this in mind, we performed a study to ascertain the lowest
catalyst loading that could be tolerated without sacrificing
enantioselection or reasonable reaction times.
Remarkably, a clear trend surfaced in that the enantiose-
lectivity of each reaction increased as the catalyst loading
decreased (Table 2). With PBAM (17) the enantioselectivity
rose from 53% ee at 20 mol % to 74% ee at just 1 mol %
loading.36 This trend was also observed using an 8-alkoxy
substituted catalyst 6,8(MeO)2PBAM (23), which gives similar
enantioselectivity to 8MeOPBAM (19). Enantioselectivity
ranged from 62% ee at 15 mol % loading to 87% ee at both
0.38 and 0.15 mol % loadings.
This feature resulted in a protocol that produced the aza-
Henry product in sufficient yield and stereoselectivity for scale-
up. 8MeOPBAM (19) was ultimately selected for further
development due to its structural simplicity and ease of
synthesis. On a 0.4 mmol scale, 2 mol % MeOPBAM (19)
catalyzed the reaction leading to 91% ee and good yield.
Enantioselectivity and yield remained unaffected as the catalyst
8
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loading was reduced to 0.5 mol %, and the reaction was
Scheme 4. Largest Scale Aryl Nitromethane Addition
concentrated to 0.4 M (Table 3, entries 2−5). Higher
Table 3. Aryl Nitromethane Addition Scale-up/Optimization
a
b
Table 4. Reduction Attempts
entry
mol %
mmol
conc (M)
ee (%)
yield (%)
1
2
3
4
5
2
0.4
0.4
0.4
0.4
0.4
2
0.1
0.1
0.2
0.4
0.4
0.4
0.4
0.4
0.4
91
82
1
92
78
1
92
84
1
91
84
0.5
0.5
0.5
0.5
0.5
91
81
c
6
77−94
86
81−90
86
d
7
2
e
conditions
yield (%)
8
20
25
89
90
e
9
89
89
CoCl2, NaBH4, MeOH, rt, 2 h
Zn/HCl, EtOH, 1.5 h
66
a
60
Enantiomer ratios of isolated material measured by HPLC using
b
Pd/C, HCO2NH4, MeOH, rt, 24 h
Raney Ni, HCO2NH4, MeOH, rt, 24 h
no rxn
no rxn
chiral stationary phases. Isolated yield of single diastereomer (200:1
dr by HPLC) after filtration/washing. Results of four runs. Imine
added in 0.2 equiv increments. Imine added in 0.1 equiv increments.
c
d
e
whereas hydrogenations with Pd/C and Raney nickel failed to
convert to product.
concentrations could not be stirred effectively as the product
precipitated from solution. Unfortunately, some initial reactions
run on larger scale (2 mmol, Table 3, entry 6) were less
enantioselective and variability was observed. Investigations
were performed on the purities of the imine, nitroalkane, and
catalyst as well as the rate of stirring, but the reason for the low
stereoselectivity remained elusive.
Ultimately, slow addition of the imine to the stirring solution
containing catalyst and nitroalkane led to consistent results
with high enantioselectivity. Five additions of 0.2 equiv of imine
led to 86% ee and 86% yield on 2 mmol scale (Table 3, entry
7). On 20 and 25 mmol scales, even slower additions (0.1 equiv
at a time) of imine led to slightly higher levels of
enantioselectivity (89% ee, Table 3, entries 8 and 9) and
good yield.
These optimized conditions produced similar results on the
largest scale attempted, over 62 mmol. Features of this reaction
include the following: 0.5 mol % catalyst loading; slow addition
of imine (∼0.06 equiv/addition over 8 h); essentially
stoichiometric amounts of the two partners (1 and 1.05
equivalents of imine and nitroalkane respectively); a relatively
high reaction concentration (0.4 M in toluene); exclusive
precipitation of the desired diastereomer from solution and
filtration to recover it; less than a day reaction time at −20 °C.
A total of 23.1 g of product 16 was produced after filtration in
91% ee and >200:1 dr. This material was then recrystallized
from toluene to provide 16 g of the desired stereoisomer
(>200:1 dr, 99% ee, Scheme 4).
With a reliable large scale preparation of the aza-Henry
adduct developed, efforts to convert this material to
(−)-Nutlin-3 commenced. The initial yield published for the
NaBH4 reduction of the nitro compound on small scale was
relatively low (66%, Table 4). Other common reduction
methods were evaluated with mixed results: treatment with Zn/
HCl provided the desired amine in modest yield (60%),
The CoCl2/NaBH4 reduction was eventually revisited for
optimization. No byproducts or unreacted starting material
were observed by NMR analysis of crude reaction mixtures
resulting from filtration, suggesting that material could have
been lost at some point in the workup. Ultimately, it was found
that the amine product was very insoluble, and copious
amounts of dichloromethane were required to fully dissolve and
separate the product from the filtered salts. After this discovery,
near-quantitative yields were obtained across reactions of
increasing scale. The largest scale reaction produced nearly
22 g of 24 with a 94% yield (Scheme 5).
An initial attempt to couple amine 24 to carboxylic acid 15
was made with DCC, resulting in a poor yield (35%) of the
desired amide. Additionally, DCC coeluted with the product
during chromatography. EDC was a better coupling reagent by
measures of yield and product isolation. This reaction provided
the desired product 25 (85% yield) after straightforward water
and solvent washes and filtrations.
A standard TFA-promoted Boc deprotection of 25
proceeded with excellent yield after a standard quench and
extractive workup. The resulting free amine 26 was converted
to isocyanate by treatment with 1,1-carbonyldiimidazole.
Subsequent reaction with 2-oxopiperazine (27)37 produced
28 in 99% yield. Straightforward washing with water provided
28 in sufficient purity for the final step.
The dehydrative cyclization between the pendant urea and
amide followed several precedents38,39 based on Hendrickson’s
work (Scheme 6).40,41 These examples demonstrated the
cyclization of an amide and a sulfonamide, and an amide and
a carbamate respectively. Each procedure used at least one
equivalent of Tf2O and 3 equivalents of Ph3PO.42 This is not
advantageous from many standpoints, but perhaps the most
significant problem is the complication it presents for
purification. Almqvist successfully used a polymer supported
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and 2 equiv of Tf2O, and over 17 g of the final compound 1
were produced in 89% yield after column chromatography.
We next tested whether the biological activity of synthetic 1
was comparable to commercially available reagent. Wild type
p53-expressing human melanoma cells were used to test the
ability of synthesized and commercial (−)-Nutlin-3 to block
melanoma cell growth and stabilize p53. We found that
addition of both compounds to the culture media caused an
approximate 90% decrease in the number of viable cells over
the vehicle (DMSO) control (Figure 3 top panels, compare A
Scheme 5. Optimized Closing Sequence for the Synthesis of
(−)-Nutlin-3
Figure 3. Biological activity of (A) synthesized and (B) commercial
(−)-Nutlin-3. Top panels: viability test. Hs294T melanoma cells were
incubated with 10 μM of (−)-Nutlin-3 or vehicle control for 5 days,
and viable cells were counted using the trypan blue exclusion test.
Bottom panels: accumulation of p53 protein in cell lysates after 10 μM
(−)-Nutlin-3 treatment, analyzed by Western Blotting. Hs294T cells
were subjected to the tested compounds or vehicle control for 72 h. β-
Actin was used as a loading control.
Scheme 6. Selected Applications of the Hendrickson
Dehydrative Cyclization
and B). Similarly, robust induction of p53 protein levels was
detected using either compound (Figure 3A and B, bottom
panels). These data demonstrate that (−)-Nutlin-3 prepared
using this batch procedure performs equivalently to commer-
cially available (−)-Nutlin-3 in downstream biological
applications.
CONCLUSIONS
■
In summary, a synthesis of (−)-Nutlin-3 has been developed at
the decagram scale (Scheme 7), aided by the development of
the new chiral bis(amidine) catalyst 19 that allows the key step
to proceed at higher temperature (−20 °C) than previously
required without attenuation of selectivity. The route is 13
steps (8 steps longest linear sequence) in length from readily
available starting materials. Procedures were developed in most
cases using glassware that was not rigorously dried and reagents
used as received. The overall yield (based on longest linear
sequence) is 32% from para-chloro benzaldehyde and 13%
from p-chlorotoluene. The procedure is summarized in Scheme
7 with yields from the largest scale reactions attempted. aza-
Henry product 16 can be produced at the 23 g scale with low
catalyst loading (0.5 mol %). A unique application of
Hendrickson’s dehydrative cyclization is employed in the final
step to produce the cis-imidazoline. The procedure has been
used to prepare 17 g of (−)-Nutlin-3 in a single batch, with
Ph3PO allowing the imidazoline products to be isolated without
the use of column chromatography.39 Despite the apparent
reusability of the polymer supported reagents, its cost is quite
prohibitive for its use on large scale.43 Our attention therefore
focused on improvement of the original Hendrickson
conditions. For reasons not yet thoroughly understood, the
stoichiometry could not be further optimized without
sacrificing conversion. The reaction mixtures in all cases,
however, were largely devoid of side products. This protocol
was superior to alternatives surveyed (e.g., SOCl2, POCl3) by
measures of both conversion and selectivity. Optimized
conditions for the dehydration employed 4 equiv of Ph3PO
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and δ 77.0 (CDCl3). IR data are reported in wavenumbers (cm−1).
Compounds were analyzed as neat films on a NaCl plate (trans-
mission). Mass spectra were recorded by use of the ionization method
noted and detected by TIC or TOF analysis. The data for all
compounds previously reported matched data previously reported in
the literature.
Scheme 7. Overall Asymmetric Synthesis of Nutlin-3
Preparation of (−)-Nutlin-3 (1). Triphenylphosphine oxide
(37.14 g, 133.4 mmol) and dry CH2Cl2 (319 mL) were added to a
1 L round-bottomed flask with a stir bar and stirred for 20 min in an
ice−water bath under argon. Tf2O (11.2 mL, 66.7 mmol) was added,
and the reaction was stirred for 20 min. The urea (20.00 g, 33.36
mmol) was added as a solid, and ∼5 mL of CH2Cl2 was used to rinse
the solid from the sides of the flask into the reaction mixture. The
mixture was stirred in the ice−water bath for 1.5 h. The mixture was
slowly quenched with satd aq NaHCO3 (325 mL, added over 10 min).
The flask was removed from the ice−water bath, stirred for 5 min
while warming to room temperature, and transferred to a 1 L
separatory funnel. The organic layer was removed, and the aqueous
layer was extracted twice with CH2Cl2. The combined organics were
dried, filtered, and concentrated. NMR analysis of this solid indicated a
4.2:1 ratio of Ph3PO to product. The crude product was purified by
column chromatography (0−4% MeOH/CH2Cl2) to provide 17.26 g
(89%) of (−)-Nutlin-3.
1-(Bromomethyl)-4-chlorobenzene (7). Note: This benzyl
bromide is a potent lachrymator. This reaction should be carried out
and worked up in a well-ventilated fume hood. The chlorotoluene (110.0
g, 868.8 mmol) and carbon tetrachloride (500 mL) were added to a 1
L round-bottomed flask containing a stir bar. N-Bromosuccinimide
(140.6 g, 789.8 mmol) was added, and the reaction was heated to
reflux temperature. AIBN (25.0 mg, 150 μmol) was added. After 10
min, AIBN (25.0 mg, 150 μmol) was added. After another 10−15 min,
the reaction began boiling vigorously. At this point, the reaction flask
was removed from heat and it continued to boil for 15 min without
external heat. When the reaction mixture stopped refluxing, the flask
was again heated to reflux temperature and AIBN (25.0 mg, 0.15
μmol) was added. The rate of reflux did not increase after the last
addition. After 15 min, the heat source was removed. A small aliquot
was concentrated and found to be >95% conversion by 1H NMR. The
reaction mixture was filtered through a glass frit and the filtered
succinimide was rinsed on the frit with hexanes. The filtrate was
concentrated (rotavap) and transferred to a 200 mL evaporation flask.
After its concentration, the mixture was allowed to sit undisturbed
overnight, resulting in crystallization of a solid out of the slightly
golden solution. The flask was placed on ice for 1 h before decanting.
The wet solid left in the flask was subjected to high vacuum for several
hours. This solid (118.1 g) was found to be a >20:1 mixture of desired
monobrominated to undesired dibrominated product, with a slight
trace of toluene starting material. The decanted liquid was subjected to
high vacuum briefly before placing on dry ice to promote
crystallization. This material was warmed to near room temperature
(10−20 °C) and filtered through a Buchner funnel. The crystalline
material was dried for several minutes on the filter before subjection to
high vacuum (∼30 min). This produced an additional 9.1 g of a white
crystalline solid that was of essentially the same purity. Overall, the
product (127.2 g, 78% yield) was produced in sufficient purity for the
next step.
column chromatography necessary after only three of the steps
(8, 2-oxopiperazine (27), and Nutlin-3).
Although the cost of (−)-Nutlin-3 has dropped 50% since
our initial disclosure, the 17 g prepared by this procedure
represents a current commercial value of ∼US $0.5 million.
Efforts to broaden the procedure to include additional novel cis-
imidazoline derivatives are underway and will be reported in
due course.
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were commercial
grade and were used as received except when noted otherwise.
Dichloromethane and tetrahydrofuran was dried by passage through a
column of activated alumina as described by Grubbs.44 Organic
extracts were dried using MgSO4 unless otherwise noted. Thin layer
chromatography (TLC) was performed using glass-backed silica gel
(250 μm) plates and flash chromatography utilized 230−400 mesh
silica gel. UV light, and/or the use of potassium permanganate
solutions were used to visualize products. Microwave reactions were
performed in a Biotage Initiator EXP US 355302 microwave reactor in
a sealed vial, at the “normal” absorption level. The temperature of
microwave reactions were monitored by an external sensor.
1-Chloro-4-(nitromethyl)benzene (8).45 Note: This benzyl
bromide is a potent lachrymator. This reaction should be carried out
and worked up in a well-ventilated fume hood. NaNO2 (40.3 g, 584
mmol), urea (46.8 g, 778 mmol), and DMF (589 mL) were added to a
1 L round-bottomed flask containing a stir bar and stirred until nearly
dissolved. Large chunks of suspended solid were chopped up by hand
with a spatula. The reaction mixture was chilled in a dry ice/acetone
bath until the internal temperature was −50 °C. Bromide (80.0 g, 389
mmol, purity as described from previous reaction) was added. 5−10
mL DMF was used to rinse residual bromide into the reaction flask.
The reaction was transferred to a −20 °C freezer and stirred for 4 h,
10 min. The flask was removed from the freezer and quickly poured
onto ∼2 L ice water in a 4 L separatory funnel. The suspension was
quickly shaken before extraction (3 × diethyl ether, ∼400 mL each).
Nuclear magnetic resonance spectra (NMR) were acquired on a
400, 500, or 600 MHz instrument. Chemical shifts are measured
relative to residual solvent peaks as an internal standard set to δ 7.26
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The combined ether extracts were dispensed into a 2 L Erlenmeyer
flask containing phloroglucinol (39.2 g, 311 mmol). This suspension
was stirred overnight (within minutes of stirring the suspension turned
to a deep red color). The suspension was poured into a 4 L separatory
funnel. The Erlenmeyer flask was rinsed with ether twice to transfer
the residual material into the separatory funnel. Approximately 1.5 L of
water was poured into the separatory funnel, and the aqueous layer
was separated without shaking to avoid emulsion. The organics were
washed three times with water (∼1.5 L each) and dried over MgSO4.
After filtration, the dried organics were concentrated by rotavap to a
red oil. This material was found to be a 53:40:7 mixture (desired nitro
compound: corresponding benzyl alcohol/dibromotoluene from
round-bottomed flask with a stir bar. The flask was then flame-dried
under vacuum and backfilled with argon. The carboxylic acid (10.00 g,
59.47 mmol) and DMF (297 mL) were added to the flask, and the
mixture was stirred at rt for 15 min. Isopropyl bromide (22.3 mL, 238
mmol) was added to the flask. The flask was purged and backflushed
with argon before placing into a sand bath. The reaction was stirred
and gradually heated from room temperature to reflux temperature
over 1.5 h. The reaction was refluxed for 4 h before removing the heat
source and cooling to 30 °C. Potassium iodide (987.3 mg, 5.947
mmol) was added to the flask. The mixture was cooled to room
temperature and stirred for 18 h. The mixture was then quenched with
3 M aq HCl and extracted three times with diethyl ether. The
combined organics were washed with 2 M aq sodium carbonate and
water, dried (MgSO4), filtered, and concentrated. The product was
1
starting material) by H NMR. This residue was purified by column
chromatography (0−1−2−3% ethyl acetate/hexanes) to obtain a
slightly golden low-melting crystalline solid (27.9 g, 42%). This
material was found to contain ∼2% of the corresponding aldehyde but
1
relatively pure by H NMR containing only a trace of monoalkylated
product. The material was purified by column chromatography (0−5%
ethyl acetate in hexanes). A bronze oil resulted (13.22 g, 93%) that was
not noticeably purer than the crude compound by 1H NMR. All
characterization data matched that in the literature.
1
was otherwise pure by H NMR.
tert-Butyl ((4-Chlorophenyl)(phenylsulfonyl)methyl)-
carbamate (11). Aldehyde (45.0, 320 mmol), carbamate (25.0 g,
214 mmol), and the sodium salt (70.1 g, 427 mmol) were combined in
a 1 L round-bottomed flask containing a sufficiently large stir bar.
MeOH (202 mL) and water (404 mL) were added, and the reaction
mixture was stirred. Formic acid (16.1 mL, 427 mmol) was added to
the reaction and the mixture continued to stir vigorously. After 9 days,
the reaction was filtered through a Buchner funnel. The contents of
the reaction flask were rinsed with diethyl ether and poured onto the
filter. The white solid was dried on the filter and transferred to a 500
mL Erlenmeyer flask. The solid was triturated with diethyl ether
(∼300 mL). The triturated suspension was filtered through a Buchner
funnel and dried. This trituration, filtration, and drying sequence was
repeated twice. The resulting solid was transferred to a 250 mL round-
bottomed flask and subjected to high vacuum for ∼2 h. This provided
53.7 g of a white, fluffy solid. 1H NMR indicated that this solid
contained a trace (<1%) of aldehyde but was otherwise very pure. The
combined filtrates were subjected to a stream of air to evaporate the
majority of the diethyl ether and transferred back to the 1 L reaction
flask (rinsed with a minimal amount of ether to fully transfer). A
stream of air was blown through the reaction flask to remove ether
before stirring vigorously. After 10 additional days, the contents of the
reaction flask were filtered through a Buchner and dried by the passage
of air through the filter. The filtered solid was subjected to the
trituration, filtration, and drying sequence (as described previously, but
using less ether) three times. The dried solid was transferred to a 100
mL round-bottomed flask and dried under high vacuum for several
hours. This produced an additional 15.2 g of white, fluffy solid that was
2-Isopropoxy-4-methoxybenzoic Acid (15).3 The ester (13.20
g, 52.32 mmol) was dispensed into a 500 mL round-bottomed flask
with a stir bar. EtOH (145 mL), water (29 mL), and KOH (8.805 g,
157.0 mmol) were added to the flask. A condenser was attached to the
flask, and the flask was placed into an oil bath (45 °C). The reaction
contents were stirred as the temperature of the bath gradually
increased to 95 °C over two hours. After 3.5 h of stirring at 95 °C, the
flask was removed from the heating bath and allowed to cool to
ambient temperature. The reaction was concentrated by rotavap to
remove most of the ethanol. The solution was diluted with water, and
3 M aq HCl was added until full precipitation was observed (pH was
checked to be ∼1). The solution was extracted three times with diethyl
ether, and the combined organics were dried over MgSO4, filtered, and
concentrated to an off-white solid (10.39 g, 94%). The compound was
1
found to be pure by H NMR.
tert-Butyl (1R,2S)-1,2-Bis(4-chlorophenyl)-2-nitroethylcarba-
mate (16). Nitroalkane (11.27 g, 65.71 mmol) and catalyst (177 mg,
313 μmol) were dispensed into a 500 mL round-bottomed flask
containing a stir bar (5 cm length). Toluene (300 mL) was added to
the flask, and the mixture was stirred at room temperature until
homogeneous. In a separate vial, imine (15.00 g, 62.58 mmol) was
dissolved in toluene (25 mL). The reaction flask was then placed into a
dry ice/acetone bath. When the stirring reaction mixture was <−40
°C, 2 mL of the imine solution was added to the reaction. The flask
was then moved into a −20 °C freezer, and stirring was continued.
Aliquots (2 mL) of the imine solution were added at approximately 30
min intervals while stirring in the −20 °C freezer. After the final
addition, the reaction was allowed to stir an additional 15 h (22 h total
reaction time) at −20 °C. The reaction flask was quickly removed
from the freezer and filtered through a Buchner funnel (12.5 cm
diameter). The flask was rinsed with ∼100 mL of hexanes to transfer
the remaining solid to the filter. The solid was rinsed with 150 mL of
cold toluene (4 °C) and 150 mL of hexanes (room temperature)
before drying for 15 min by the passage of air through the filter. The
solid was transferred to a 250 mL round-bottomed flask and dried
under vacuum for 2h. The crude solid (23.07 g, 90%) was found to be
200:1 dr, 91% ee by chiral HPLC. The material was transferred to a 2
L Erlenmeyer flask and toluene (1.13 L, 49 mL/gram of crude
product) was added to the flask. The suspension was swirled while
being heated in a 70 °C water bath until fully dissolved. The flask was
then placed on the benchtop to cool. After 130 min, the crystallized
suspension was filtered through a Buchner funnel into a 2 L filter flask.
The crystallized material was dried on the filter for at least 15 min
before transfer to a 250 mL round-bottomed flask and subjection to
vacuum for 2 h. This produced 12.49 g of crystallized product that was
found to be 200:1 dr, 99% ee by chiral HPLC. The filtrate from this
recrystallization was redissolved with heat in a 70 °C water bath and
left to cool to room temperature and age 19 h. The mother liquor was
separated from the newly formed globular crystals by filtration through
fluted filter paper into a 2 L Erlenmeyer flask. This filtrate was placed
in an ice water bath for 1 h. The contents of the flask were filtered
1
found to be equally pure as the first sulfone batch by H NMR. The
combined filtrates were again transferred back to the reaction flask and
concentrated by a stream of air as described above. The mixture was
stirred for an additional 25 days. After implementation of the same
filtration and trituration sequence, 3.4 g of white, fluffy solid was
obtained. This material was found to be of the same purity as previous
1
batches by H NMR. Overall, 72.4 g (89%) of sulfone was isolated.
This material was sufficiently pure for use in the next step.
(E)-tert-Butyl 4-Chlorobenzylidenecarbamate (12). Potassium
carbonate (64.59 g, 467.3 mmol) and sodium sulfate (75.85 g, 534.0
mmol) were loaded into a 1 L three-neck round-bottomed flask with
stir bar. The flask was flame-dried, backfilled with argon, and attached
to a condenser. Sulfone (25.49 g, 66.75 mmol) was then added
through a side neck before dry THF (556 mL) was added via cannula.
The flask was place into a preheated oil bath and refluxed for 6 h. The
flask was allowed to cool to room temperature before filtering through
a glass frit. Ether was used to rinse residual solid from the reaction
flask and the solid on the filter. The filtrate was concentrated by
rotavap before subjection to high vacuum. White solid (15.18 g, 95%)
was produced that was found to contain 4% of the corresponding
1
product but otherwise pure by H NMR. All characterization data
matched that in the literature. This material was used in the next
reaction without further purification.
Isopropyl 2-Isopropoxy-4-methoxybenzoate (14).46 Potassi-
um carbonate (32.86 g, 237.9 mmol) was dispensed into a 500 mL
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through a Buchner funnel (8.5 cm diameter). After the crystalline
material was dried on the filter by the passage of air, it was transferred
to a 250 mL round-bottomed flask and placed under high vacuum for
3 h. This produced 3.46 additional grams of product that was found to
be 200:1 dr and 99% ee. The combined yield from the
recrystallizations was 15.98 g (62%) of diastereo- and enantiomerically
pure compound.
light brown solid. This material was dissolved in dichloromethane and
then washed with 3 M aq NaOH. The combined organic layers were
dried over MgSO4 and concentrated to afford a brown/red powder
(100.9 mg, 72%): mp 112.0−114.0 °C; [α]2D0 +274 (c 1.03, CHCl3); Rf
= 0.14 (10% MeOH/0.5% AcOH/CH2Cl2); IR (film) 3257, 2930,
1
2858, 1607, 1538 cm−1; H NMR (400 MHz, CDCl3) δ 6.83 (d, J =
2.4 Hz, 2H), 6.58 (d, J = 2.4 Hz, 2H), 5.71 (br s, 2H), 5.60 (s, 2H),
4.05−3.92 (m, 2H), 3.97 (s, 2H), 3.82 (s, 6H), 3.34−3.23 (m, 2H),
3.23−3.12 (m, 4H), 2.35−2.24 (m, 2H), 1.93−1.82 (m, 8H), 1.82−
1.74 (m, 2H), 1.55−1.35 (m, 4H); 13C NMR (100 MHz, CDCl3) ppm
156.5, 154.4, 153.6, 152.6, 136.7, 119.0, 100.2, 96.8, 94.4, 56.3, 56.2,
55.3, 51.6, 33.1, 25.4, 24.9; HRMS (CI) exact mass calcd for
C36H46N6O4 [M]+ 626.3575, found 626.3550.
8MeOPBAM (19). A 0.5−2.0 mL microwave vial was charged with
4
the corresponding ClBAM (200.0 mg, 402.1 μmol), pyrrolidine (132
μL, 1.61 mmol), and trifluoromethylbenzene (1.2 mL). This
suspension was heated at 180 °C and stirred in the microwave for
30 min. The reaction mixture was purified by column chromatography
(2−5% methanol in dichloromethane with 0.5% AcOH) to provide a
light brown solid. This material was dissolved in dichloromethane and
then washed with 3 M aq NaOH. The combined organic layers were
dried over MgSO4 and concentrated. The resulting solid was dissolved
in EtOAc and washed with water, dried over MgSO4, filtered, and
concentrated to a light brown solid (108.4 mg, 48%): [α]2D0 +320 (c
0.14, CHCl3); Rf = 0.24 (10% MeOH/CH2Cl2); IR (film) 3244, 2933,
tert-Butyl (1R,2S)-2-Amino-1,2-bis(4-chlorophenyl)-
ethylcarbamate (24). The nitroalkane (25.00 g, 60.78 mmol) was
dispensed into a 2 L three-neck round-bottomed flask with stir bar,
and MeOH was added (500 mL) before the mixture was stirred at
room temperature. CoCl2 (7.891 g, 60.78 mmol) was added, and the
reaction mixture was chilled (0 °C). A septum was attached to each of
the three necks, and a needle attached to an empty balloon was
inserted into each septum. NaBH4 (11.50 mg, 303.9 mmol) was slowly
added over a 1 h period. The reaction mixture was stirred at 0 °C for
an additional 1 h (starting material consumed by TLC) before the
mixture was quenched with 325 mL of water, 550 mL of 1 M aq HCl,
and 375 mL of satd aq NH4OH. The pH of the reaction mixture was
found to be ∼10. The mixture was filtered through a Buchner funnel
(17 cm diameter). Water (600 mL) was used to rinse the flask and
wash the filtered solid. The solid was dried on the filter for 15 min.
The filtered flask was changed before rinsing the filtered solid with
CH2Cl2 (950 mL in several portions). This CH2Cl2 filtrate was
transferred to a 1 L separatory funnel and the water was separated.
The organic layer was dried, filtered, concentrated by rotavap, and
subjected to high vacuum (5 h). This produced a white solid (21.74 g,
1
2857, 1637, 1593 cm−1; H NMR (400 MHz, CDCl3) δ 7.49 (d, J =
8.4 Hz, 2H), 6.93 (dd, J = 7.6, 7.6 Hz, 2H), 6.88 (d, J = 7.2 Hz, 2H),
5.78 (br s, 2H), 5.52 (br s, 2H), 4.10−4.00 (m, 2H), 4.00 (s, 6H),
3.41−3.29 (m, 4H), 3.28−3.15 (m, 4H), 2.40−2.27 (m, 2H), 1.90−
1.73 (m, 8H), 1.73−1.65 (m, 2H), 1.60−1.40 (m, 4H); 13C NMR
(150 MHz, CDCl3, 325 K) ppm 157.5, 153.8, 153.7, 141.6, 119.5,
118.5, 117.3, 109.0, 93.3, 56.5, 56.3, 51.7, 33.1, 25.4, 24.9; HRMS
(ESI) exact mass calcd for C34H43N6O2 [M + H]+ 567.3448, found
567.3431.
2,4-Dichloro-8-methoxyquinoline (21). Phosphorus(V) oxy-
chloride (50 mL) was added through a running condenser into a
three-neck round-bottom flask equipped with a stir bar containing
malonic acid (13.0 g, 125 mmol) at room temperature. While the
mixture was stirring, o-anisidine (14.1 mL, 125 mmol) was added in
small portions over a period of 15 min through an open neck of the
round-bottom flask. The reaction mixture was heated and stirred at
reflux for 7 h. The reaction mixture was allowed to cool to room
temperature before it was poured over crushed ice. Soxhlet extraction
of the crude solid with hexanes for ∼24 h followed by cooling of the
obtained extract yielded a yellow solid precipitate (2.6135 g, 9%) that
1
94%) which was judged to be of high purity by H NMR.
tert-Butyl (1R,2S)-1,2-Bis(4-chlorophenyl)-2-(2-isopropoxy-
4-methoxybenzamido)ethylcarbamate (25). The amine (21.5 g,
56.4 mmol) and carboxylic acid (11.85 g, 56.39 mmol) were dissolved
in CH2Cl2 (500 mL) at room temperature in a 1 L round-bottomed
flask. The solution was chilled to 0 °C in an ice/water bath, and EDC
(14.05 g, 73.30 mmol) and DMAP (688 mg, 5.64 mmol) were added.
The reaction mixture was stirred vigorously (reaction thickens over
time) and allowed to gradually warm to room temperature. After 16 h,
the reaction mixture was diluted with CH2Cl2 (125 mL) and the
precipitated solid was divided and stirred with a spatula by hand. The
reaction was filtered through a Buchner funnel. The reaction flask was
rinsed and poured onto the filter with CH2Cl2 (300 mL). An
additional 300 mL of CH2Cl2 was used to wash the solid on the filter.
This solid was transferred to a 500 mL round-bottomed flask and
triturated with 300 mL CH2Cl2 by manual stirring with a spatula. The
solid was filtered through a Buchner funnel and washed out of the flask
with an additional 200 mL CH2Cl2. The solid was transferred to a 200
mL round-bottomed flask and dried under vacuum overnight. This
produced 24.4 g of white solid with some light blue color and was
determined to be an ∼6:1 mixture of product to EDC (∼4% EDC urea
1
was pure by H NMR: mp 127.5−128.5 °C; Rf = 0.08 (10% EtOAc/
1
hexanes); IR (film) 3097, 3008, 1576, 1561 cm−1; H NMR (500
MHz, CDCl3) δ 7.76 (d, J = 8.5 Hz, 1H), 7.57 (dd, J = 8.0, 8.0 Hz,
1H), 7.55 (s, 1H), 7.15 (d, J = 7.5 Hz, 1H), 4.08 (s, 3H); 13C NMR
(100 MHz, CDCl3) ppm 154.8, 148.9, 144.3, 139.9, 128.1, 126.4,
122.7, 115.6, 109.8, 56.3; HRMS (CI) exact mass calcd for
C10H7Cl2NO [M]+ 226.9899, found 226.9890.
4Cl8MeOQuinBAM (22). A 100 mL round-bottom flask was
charged with Pd(dba)2 (25.2 mg, 43.8 μmol), rac-BINAP (27.3 mg,
43.8 μmol), sodium tert-butoxide (632.0 mg, 6.576 mmol), (R,R)-
diaminocyclohexane (250.3 mg, 2.192 mmol), and the quinoline
(1.0000 g, 4.385 mmol).47 Toluene (22 mL) was added, and the
reaction mixture was stirred at 70 °C for 3.5 h. The reaction was
cooled to room temperature, diluted with CH2Cl2, and filtered through
Celite. The filtrate was concentrated and purified by column
chromatography (25−50% ethyl acetate in hexanes) to provide a
yellow solid (642.6 mg, 62%): [α]2D0 +530 (c 0.16, CHCl3); Rf = 0.31
1
by mass) by H NMR. This 24.4 g of material (solid A) was set aside
for later purification. The combined filtrates were poured into a 1 L
separatory funnel, washed three times with water, dried, and
concentrated to a solid. This solid was transferred to a 250 mL
Erlenmeyer flask and triturated with 150 mL of CH2Cl2. The solid was
filtered through a Buchner funnel and washed two times with CH2Cl2
(50 mL each) and once with hexanes (50 mL). After transfer to an
evaporation flask, the solid was subjected to high vacuum overnight.
This produced 6.87 g of white-yellow solid which contained a trace of
EDC and some other impurities. This material was then transferred to
a 125 mL Erlenmeyer flask and triturated with 50/50 CH2Cl2/hexanes
(70 mL). The solid was filtered through a Buchner funnel and washed
with 50 mL (50/50 CH2Cl2/hexanes). After transfer to a 100 mL
round-bottomed flask, the solid (solid B) was subjected to high
vacuum and set aside for later purification. The 24.4 g of material
isolated from the first filtration (solid A) was added to a 500 mL
1
(50% EtOAc/hexanes); IR (film) 3240, 2933, 1607, 1545 cm−1; H
NMR (400 MHz, CDCl3) δ 7.54 (dd, J = 8.4, 0.8 Hz, 2H), 7.17 (dd, J
= 7.6, 0.8 Hz, 2H), 7.01 (dd, J = 7.6, 0.8 Hz, 2H), 6.59 (s, 2H), 6.38
(br s, 2H), 4.15−3.95 (m, 2H), 4.04 (s, 6H), 2.45−2.30 (m, 2H),
1.85−1.70 (m, 2H), 1.50−1.30 (m, 4H); 13C NMR (150 MHz,
CDCl3) ppm 155.9, 153.2, 142.1, 140.0, 122.2, 121.9, 116.2, 112.7,
109.8, 56.6, 56.2, 32.5, 24.7; HRMS (ESI) exact mass calcd for
C26H27Cl2N4O2 [M + H]+ 497.1511, found 497.1521.
6,8(MeO)2PBAM (23). A 0.5−2.0 mL microwave vial was charged
4
with the corresponding ClBAM (125.0 mg, 224.2 μmol), pyrrolidine
(200 μL, 2.44 mmol), and trifluoromethylbenzene (600 μL). This
suspension was heated at 180 °C and stirred in the microwave for 10
min. The reaction was then concentrated and purified by column
chromatography (0−10% methanol in dichloromethane) to provide a
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Erlenmeyer flask and triturated with 400 mL of 50/50 CH2Cl2/
hexanes with the aid of manual stirring by hand. This material was
filtered by Buchner funnel and washed with hexanes. This solid was
combined with solid B in a 1 L Erlenmeyer flask and triturated by stir
bar and manual spatula stirring (dividing large, undispersed chunks) by
hand with 600 mL 50/50 CH2Cl2/hexanes. This material was filtered
(Buchner funnel, large chunks were divided on the filter) and washed
with 300 mL 50/50 CH2Cl2/hexanes. After being dried on the filter
for 1 h, the solid was transferred to a 250 mL evaporation flask and
subjected to high vacuum for 3 h. This produced 28.6 g of white solid
through a Buchner funnel. Some solid was present in the filtrate before
the filtrate was concentrated by rotavap. The material was redissolved
in 600 mL of CH2Cl2 and filtered through Celite on a glass frit. This
filtrate was concentrated to a white solid (25.5 g, 99%) that was found
1
to be pure by H NMR.
2,4-Dichloro-6,8-dimethoxyquinoline (S1). Compound S1 was
prepared using 6,8-dimethoxyaniline (10.0 g, 65.3 mmol), malonic acid
(6.79 g, 65.3 mmol), and POCl3 (26 mL) according to the procedure
for S5. The reaction time was 2 h. The crude solid was extracted
(Soxhlet) with hexanes for 3 d to provide the title compound as a
green-brown solid (1.376 g, 8%): mp 183.5−184.5 °C; Rf = 0.09 (10%
1
with only a very faint blue tint. By H NMR, this material was a 6:1
1
EtOAc/hexanes); IR (film) 3080, 2964, 1615, 1562 cm−1; H NMR
mixture of desired product to EDC urea. This solid was transferred to
a 1 L Erlenmeyer flask and triturated with a suspension of 500 mL 4:1
water:hexanes. After filtration through a Buchner funnel, the solid was
washed successively on the filter with 200 mL hexanes, 200 mL water,
and 200 mL of hexanes. The solid was dried on the filter for 20 min
before transferring to a flask and subjecting to high vacuum. This
produced 26.7 g (83%) of a white solid that was found to be a >20:1
mixture of desired product to EDC urea.
(400 MHz, CDCl3) δ 7.50 (s, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.77 (d, J
= 2.4 Hz, 1H), 4.03 (s, 3H), 3.95 (s, 3H); 13C NMR (100 MHz,
CDCl3) ppm 159.4, 155.8, 146.1, 142.7, 136.6, 127.1, 122.9, 103.0,
93.7, 56.3, 55.7; HRMS (ESI) exact mass calcd for C11H10Cl2NO2 [M
+ H]+ 258.0089, found 258.0094.
4Cl6,8(MeO)2QuinBAM (S2). A 25 mL round-bottom flask was
charged with Pd(dba)2 (22.3 mg, 38.7 μmol), rac-BINAP (24.1 mg,
38.7 μmol), sodium tert-butoxide (558.4 mg, 5.811 mmol), (R,R)-
diaminocyclohexane (221.2 mg, 1.937 mmol), and the quinoline
(1.000 g, 3.874 mmol). Toluene (13 mL) was added, and the reaction
mixture was stirred at 85 °C. The reaction mixture was monitored by
TLC, and complete conversion was observed after 65 min. The
reaction was cooled to room temperature, diluted with CH2Cl2, and
filtered through Celite. The filtrate was concentrated and purified by
column chromatography (25−30% ethyl acetate in hexanes) to
provide a yellow/orange solid (775.7 mg, 72%): mp 120.0−122.0
°C; [α]2D0 +434 (c 1.28, CHCl3); Rf = 0.09 (25% EtOAc/hexanes); IR
(film) 3245, 2934, 2856, 1606, 1545 cm−1; 1H NMR (400 MHz,
CDCl3) δ 6.86 (d, J = 2.4 Hz, 2H), 6.68 (d, J = 2.4 Hz, 2H), 6.57 (s,
2H), 6.09 (s, 2H), 4.01 (s, 6H), 3.98−3.90 (m, 2H), 3.88 (m, 6H),
2.42−2.28 (m, 2H), 1.86−1.70 (m, 2H), 1.52−1.35 (m, 4H); 13C
NMR (100 MHz, CDCl3) ppm 155.0, 154.7, 154.2, 141.2, 136.0,
122.1, 112.6, 102.2, 94.7, 56.6, 56.2, 55.4, 32.5, 24.7; HRMS (ESI)
exact mass calcd for C28H31Cl2N4O4 [M + H]+ 557.1722, found
557.1738.
N-((1S,2R)-2-Amino-1,2-bis(4-chlorophenyl)ethyl)-2-isopro-
poxy-4-methoxybenzamide (26). The amide (26.4 g, 46.0 mmol)
was dispensed into a 1 L round-bottomed flask containing a stir bar.
Dichloromethane (455 mL) was added, and the reaction mixture was
stirred at room temperature. TFA (∼38 mL) was added over a period
of 10 min via an addition funnel to the stirred suspension (after the
addition of the first ∼5 mL the mixture became homogeneous). After
stirring for 2.5 h, the reaction mixture was checked by TLC and found
to still contain starting material. Approximately 15 mL TFA (53 mL
total, 710 mmol) was added over 2 min. After stirring for 1.5 h, the
reaction mixture was found to no longer contain starting material by
TLC. The reaction flask was placed into an ice−water bath, and 300
mL of satd aq NaHCO3 was slowly added to the stirring mixture. After
transfer to a 2 L Erlenmeyer flask with 50 mL of satd aq NaHCO3 and
50 mL of CH2Cl2, the following amounts of solutions and solvents
were carefully and successively added to the vigorously stirring
mixture: 50 mL of 3 M aq NaOH, 100 mL of CH2Cl2, 50 mL of 3 M
aq NaOH, 100 mL of CH2Cl2, 200 mL of water, 100 mL of 3 M aq
NaOH, 100 mL of CH2Cl2, and 200 mL of water. The suspension was
vigorously stirred for 1.5 h. After transfer to a 4 L separatory funnel,
the organic layer was removed. The aqueous layer was extracted twice
with CH2Cl2 (400 mL each). The combined organic layers were dried,
filtered, and concentrated by rotavap and high vacuum. This produced
20.0 g (92%) of white solid that contained a trace of EDC urea and
CH2Cl2 but was otherwise pure by 1H NMR. This material was
sufficiently pure for use in the next reaction.
Biological Activity Test. A stock solution of 30 mM (−)-Nutlin-3
was prepared in DMSO. Hs294T cells were obtained from ATCC and
maintained in DMEM/F12 media supplemented with 10% fetal bovine
serum, 100U/mL penicillin, and 100ug/mL streptomycin. For
viability, test cells were incubated with 10 μM (−)-Nutlin-3 for 5
days, trypsinized, and mixed 9:1 with 0.4% buffered trypan blue. Viable
trypan blue negative cells were counted in a hemocytometer. For the
analysis of p53 protein levels, Hs294T cells were incubated with 10
μM (−)-Nutlin-3 or an equivalent volume of vehicle DMSO for 3
days. Cells lysates were analyzed by Western Blotting with p53-specific
(DO-1, Calbiochem) and β-actin-specific (Cell Signaling) antibodies.
2-Oxopiperazine (27).37 This compound was prepared according
to the patent procedure on a scale using 19.6 g of ethyl chloroacetate
(160 mmol). After a 45 h reaction time, the reaction mixture was
concentrated by rotary evaporator with water bath heating (60−70
°C). The resulting orange oil was purified by column chromatography
(3−10% MeOH in CH2Cl2 w/1% NH4OH). A yellow solid (9.9 g)
was obtained. This material was recrystallized from acetone (40 mL)
to obtain a yellow-brown solid (3.75 g, 23%) that was sufficiently pure
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all previously unreported
compounds, as well as HPLC traces for 16. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
by H NMR.
N-((1R,2S)-1,2-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-
methoxybenzamido)ethyl)-3-oxopiperazine-1-carboxamide
(28). The amine (20.30 g, 42.87 mmol) was dispensed into a 500 mL
round-bottomed flask equipped with a stir bar. Dry dichloromethane
(214 mL) was added, and the reaction mixture was stirred at room
temperature. 1,1-Carbonyldiimidazole (8.342 g, 51.45 mmol) was
added and stirring continued for 1.5 h. TLC indicated complete
consumption of starting material (Rf = 0.36 in 5% MeOH/CH2Cl2),
and a new spot had appeared (Rf = 0.43 in 5% MeOH/CH2Cl2). The
oxopiperazine (6.800 g, 67.93 mmol) was added to the reaction and
stirring continued for 18 h. TLC indicated the complete consumption
of isocyanate. The reaction was diluted with CH2Cl2 (750 mL) and
washed three times with water (500 mL each) in a 4 L separatory
funnel. After the last wash was shaken, 200 mL of brine was added to
alleviate an emulsion in the aqueous layer. The combined organic
layers were dried over MgSO4 in a 2 L Erlenmeyer flask and filtered
AUTHOR INFORMATION
Corresponding Author
*E-mail: jeffrey.n.johnston@vanderbilt.edu.
■
Present Address
†Department of Chemistry, Colorado State University, Fort
Collins, CO 80523.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health
(NIH): GM084333 (J.N.J.), CA116021-S1 (A.R.), K12-
■
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Article
(14) Fotouhi, N.; Haley, G. J.; Simonsen, K. B.; Vu, B. T.; Webber, S.
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110996, Nov 24, 2005.
CA90625 (A.E.V.), and the TVHS, Department of Veterans
Affairs (A.R.). We are grateful to Brandon Vara for assistance
with purification of the final compound.
(15) Kornblum, N.; Larson, H. O.; Blackwood, R. K.; Mooberry, D.
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